Predictive assay for immune response

ABSTRACT

The present invention relates to an in vitro method for determining the ability of a vaccine composition which comprises one or more antigens or a nucleic acid molecule which encodes one or more antigens to stimulate a T cell response. In one embodiment, the method comprises the steps of: (1) contacting antigen presenting cells in culture with a vaccine composition selected from among the group of vaccine compositions, thereby, if one or more of the antigens or nucleic acid molecules can be taken up and processed by the antigen presenting cells, producing one or more processed antigens; (2) contacting the antigen presenting cells with T cells under conditions sufficient for the T cells to respond to one or more of the processed antigens; (3) determining whether the T cells respond to one or more of the processed antigens; whereby if the T cells respond to one or more of the processed antigens, then the vaccine composition stimulates a T cell response; and (4) repeating steps (1), (2) and (3) with each vaccine composition in the group, thereby identifying vaccine compositions which stimulate a T cell response; and, if one or more of the vaccine compositions stimulates a T cell response, (5) selecting at least one vaccine composition which stimulates a T cell response for assessment in one or more animals and/or human subjects.

RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.09/159,172, filed Sep. 23, 1998. The entire teachings of the aboveapplication(s) are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Among the greatest successes in the field of public health is widespreadvaccination against a variety of formerly common infectious diseases.For example, public vaccination programs in the United States haveeradicated smallpox and dramatically reduced the incidence of diseasessuch as measles, rubella, polio and diphtheria, among others. However,the development of novel vaccine compositions is still an active area ofresearch. In particular, the development of effective vaccines for anumber of diseases for which no clinically proven vaccine exists remainsan important goal. For example, a vaccine which protects againstinfection by human immunodeficiency virus (HIV) is a primary goal inefforts to control the spread of AIDS. Also needed are vaccinecompositions which have improved efficacy in comparison to vaccines incurrent use.

The efficacy of a vaccine for use in humans depends upon the ability ofthe vaccine formulation to elicit an immune response which is sufficientto provide protection against subsequent challenge with the pathogen.Experimental vaccines are typically evaluated first in vivo in smallanimals, such as mice, guinea pigs or rabbits. The assessment of theexperimental vaccines generally relies upon measurements of serumantibody responses and, sometimes, antigen-specific lymphocyteproliferative responses. Vaccine formulations which are successful inthese animal models are then tested in sub-human primates and, finally,in humans.

The assessment of a test vaccine in an animal model is costly and takesconsiderable time. Typically, several doses of vaccine are administeredto the animal at intervals of several weeks. The immune response ofprimates to a given test vaccine is often less than that of smalleranimals, and clinical studies in humans are ultimately required todetermine the efficacy of a test vaccine. In addition to the large costsassociated with purchasing and housing animals for long periods of time,each step of the process requires a minimum of several months. Thus, thenumber of experimental vaccines which can be evaluated using prior artmethods is necessarily limited, with the possible result thatpotentially useful vaccine formulations may never be tested.

There is, therefore, a need for an in vitro test for determining thehuman immune response to an experimental vaccine construct which wouldallow the rapid evaluation of large numbers of candidate vaccinecompositions within a short time period and at reasonable cost.

SUMMARY OF THE INVENTION

The present invention relates to a method for assessing the ability of acandidate vaccine composition to stimulate a T cell response. In oneembodiment, the invention provides a method for selecting one or morevaccine compositions from among a group of vaccine compositions for invivo assessment, for example, in one or more animal or human subjects.Each of the vaccine compositions comprises one or more antigens or oneor more nucleic acid molecules encoding one or more antigens. The methodcomprises the steps of: (1) contacting antigen presenting cells inculture with a vaccine composition selected from among the group ofvaccine compositions, thereby, if one or more of the antigens or nucleicacid molecules can be taken up and processed by the antigen presentingcells, producing one or more processed antigens; (2) contacting theantigen presenting cells with T cells under conditions sufficient forthe T cells to respond to one or more of the processed antigens; (3)determining whether the T cells respond to one or more of the processedantigens; whereby if the T cells respond to one or more of the processedantigens, then the vaccine composition stimulates a T cell response; and(4) repeating steps (1), (2) and (3) with each additional vaccinecomposition in the group, thereby identifying the vaccine compositionswithin the group which stimulate a T cell response; and, if one or moreof these vaccine compositions stimulates a T cell response, (5)selecting at least one vaccine composition which stimulates a T cellresponse for assessment in one or more animals and/or in one or morehuman subjects.

In another embodiment, the invention relates to a method of selecting avaccine composition from a group consisting of two or more vaccinecompositions for assessment in one or more animals or in one or morehuman subjects. Each of the vaccine compositions comprises one or moreantigens or one or more nucleic acid molecules encoding one or moreantigens. The method comprises the steps of: (1) contacting antigenpresenting cells in culture with a vaccine composition selected fromamong said group of vaccine compositions, thereby, if one or more of theantigens or nucleic acid molecules are taken up and processed by theantigen presenting cells, producing one or more processed antigens; (2)contacting the antigen presenting cells with T cells under conditionssufficient to produce a T cell response to one or more of the processedantigens, thereby producing a vaccine composition-stimulated T cellresponse; (3) measuring the vaccine composition-stimulated T cellresponse; (4) repeating steps (1), (2) and (3) with each of theremaining vaccine compositions in the group, thereby identifying thevaccine composition or compositions which stimulate the greatest T cellresponse; (5) selecting the vaccine composition or compositions whichstimulate the greatest T cell response for assessment in one or moreanimals and/or in one or more human subjects.

In a further embodiment, the invention relates to a method for assessingthe ability of a vaccine composition comprising one or more antigens orone or more nucleic acid molecules encoding one or more antigens tostimulate a protective T cell response. The method comprises the stepsof: (1) contacting human antigen presenting cells in culture with thevaccine composition, thereby, if one or more of the antigens or nucleicacid molecules can be taken up and processed by the antigen presentingcells, producing one or more processed antigens; (2) contacting theantigen presenting cells with human T cells under conditions sufficientto produce a T cell response to one or more of the processed antigens,thereby producing a T cell response; (3) measuring the T cell response;and, if the T cell response is greater than a pre-selected value, (4)assessing the ability of the vaccine composition to stimulate aprotective T cell response in one or more animals or in one or morehuman subjects.

In another embodiment, the method of the invention comprises the stepsof: (1) contacting human antigen presenting cells in culture with thevaccine composition, whereby, if one or more of the antigens are takenup and processed by the antigen presenting cells, said antigen orantigens are processed by the antigen presenting cells, therebyproducing one or more processed antigens; (2) contacting the antigenpresenting cells of step (1) with human T cell clones which are specificfor an epitope within one or more of the antigens for a period of timesufficient for the human T cell clones to respond to one or more of theprocessed antigens; and (3) determining whether the human T cell clonesrespond to the processed antigen or antigens. If the T cell clonesrespond to the processed antigen or antigens, the method can,optionally, further include the step of assessing the vaccinecomposition in one or more animals or human subjects.

Preferably, the vaccine composition includes at least one antigen whichcomprises a T cell epitope, and the T cells are T cell clones which arespecific for a T cell epitope in at least one of the antigens. In oneembodiment, the T cells are CD8⁺ T cells and the vaccine compositionincludes at least one antigen comprising antigen a CD8 epitope. In thisembodiment, the T cell response to the processed antigen can be, forexample, T cell proliferation, cytolysis of the antigen presenting cellsor the production of one or more cytokines.

In another embodiment, the T cells are CD4⁺ T cells and the vaccinecomposition includes at least one antigen which comprises a CD4 epitope.In this embodiment, the T cell response to the processed antigen whichis determined can be, for example, T cell proliferation, stimulation ofantibody production by B cells or production of one or more cytokines.

The present invention offers several advantages over prior art methodsof evaluating candidate vaccine compositions. For example, the method ofthe invention can be completed in a relatively short time period. Thepresent method can also be used as a first screen to determine whichcandidate compositions should be evaluated in much more expensive andtime consuming in vivo tests. Thus, the method of the invention enablesthe efficient and cost effective evaluation of large numbers ofpotential vaccine compositions, increasing the possibility thateffective vaccine compositions will be discovered.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph showing the increase in percent lysis againstinfluenza virus strain A/Texas compared to day 0 for several fluzoneformulations at day 14.

FIG. 1B is a graph showing the increase in percent lysis againstinfluenza virus strain A/Texas compared to day 0 for several fluzoneformulations at day 56.

FIG. 2A is a graph showing the increase in percent lysis againstinfluenza virus strain A/Johannesburg compared to day 0 for severalfluzone formulations at day 14.

FIG. 2B is a graph showing the increase in percent lysis againstinfluenza virus strain A/Johannesburg compared to day 0 for severalfluzone formulations at day 56.

DETAILED DESCRIPTION OF THE INVENTION

Successful vaccines deliver to a host one or more antigens derived froma pathogen, thereby stimulating an immune response which protectsagainst subsequent challenge with the pathogen. Such vaccines can take avariety of forms, including attenuated or killed pathogens, for example,viruses or bacteria; one or more proteins or peptides derived from apathogen or synthetic or recombinant versions of such proteins orpeptides; or one or more nucleic acid molecules encoding one or moreproteins or peptides from the pathogen, such as a naked DNA vaccine or anucleic acid molecule administered in a suitable vector, such as arecombinant virus or bacterium or an immunostimulating complex. Vaccinesagainst cell proliferative diseases, such as cancers, typically utilizeproteins or fragments thereof, or nucleic acid molecules encodingproteins or fragments thereof, which are unique to diseased cells orgenerally more abundant in diseased cells compared to healthy cells.

Cell-mediated immunity is dependent upon lymphocytes known as B cellsand T cells. B cells produce antibodies targeted against extracellularantigens. T cells recognize antigen fragments (peptides) which aredisplayed at the surface of a host cell. Such antigen fragments resultfrom uptake of the antigen by a host cell, or synthesis of the antigenwithin the host cell, followed by cleavage of the antigen within thecell. Although it is probable that most successful vaccines elicit bothT cell and B cell responses, current methods for evaluating testvaccines generally focus on antibody production by B cells, and do notassess the ability of the test vaccine to elicit a T cell response.

Foreign proteins which are synthesized within the host cell or are takenup by the host cell via specific receptors are fragmented within thecytosol of the cell. One or more of the resulting peptides can becomeassociated with class I major histocompatibility molecules (MHC I), andthe resulting complexes are then presented at the surface of the cell.These MHC I/peptide complexes are recognized by specific T cellreceptors in certain CD8⁺ T cells, and the peptides so presented arereferred to as CD8 epitopes.

A foreign protein can be taken up by a host cell nonspecifically viaendocytosis and then fragmented into peptides in a cellular lysosomal orendosomal compartment. One or more of these peptides can then becomeassociated with a class II major histocompatibility molecule (MHC II) toform a complex which is then presented at the surface of the host cell.These MHC II/peptide complexes are recognized by CD4⁺ T cells expressinga specific receptor which recognizes the MHC I/peptide complex. Thesepeptides are referred to as CD4 epitopes.

Peripheral T cells in the blood and organs of the immune system (e.g.spleen and lymph nodes) exist in a quiescent or resting state. Uponinteraction of T cells with an MHC/epitope complex, the T cellsproliferate and differentiate into activated cells having a variety offunctions. CD8⁺ T cells typically become cytotoxic upon activation anddestroy antigen-presenting cells via direct contact. Activated CD4⁺ Tcells provide a helper function to B cells, enabling B cells todifferentiate into antibody-producing cells. Activated CD8⁺ T cells andCD4⁺ T cells release a variety of cytokines (lymphokines orinterleukins), which can, for example, control differentiation of manyclasses of lympholytic precursor cells.

In one embodiment, the invention provides a method for selecting one ormore vaccine compositions from among a group of two or more vaccinecompositions for in vivo assessment in one or more animals and/or humansubjects. Each of the vaccine compositions comprises one or moreantigens or one or more nucleic acid molecules encoding one or moreantigens. The method comprises the steps of: (1) contacting antigenpresenting cells in culture with a vaccine composition selected fromamong said group of vaccine compositions, thereby, if one or more of theantigens or nucleic acid molecules are taken up and processed by theantigen presenting cells, producing one or more processed antigens; (2)contacting the antigen presenting cells with T cells under conditionssufficient for the T cells to respond to one or more of the processedantigens; (3) determining whether the T cells respond to one or more ofthe processed antigens; whereby if the T cells respond to one or more ofthe processed antigens, then the vaccine composition stimulates a T cellresponse; and (4) repeating steps (1), (2) and (3) with each vaccinecomposition in the group, thereby identifying vaccine compositions whichstimulate a T cell response; and, if one or more of the vaccinecompositions stimulates a T cell response, (5) selecting at least onevaccine composition which stimulates a T cell response for assessment invivo.

In another embodiment, the invention relates to a method of selecting atleast one vaccine composition from a group consisting of two or morevaccine compositions for assessment in one or more animals and/or humansubjects. Each of the vaccine compositions comprises one or moreantigens or one or more nucleic acid molecules encoding one or moreantigens. The method comprises the steps of: (1) contacting antigenpresenting cells in culture with a vaccine composition selected fromamong said group of vaccine compositions, thereby, if one or more of theantigens or nucleic acid molecules can be taken up and processed by theantigen presenting cells, producing one or more processed antigens; (2)contacting the antigen presenting cells with T cells under conditionssufficient to produce a T cell response to one or more of the processedantigens, thereby producing a vaccine composition-stimulated T cellresponse; (3) measuring the vaccine composition-stimulated T cellresponse; (4) repeating steps (1), (2) and (3) with each of theremaining vaccine compositions in the group, thereby identifying one ormore vaccine compositions which stimulate the greatest T cell response;and (5) selecting the vaccine composition or compositions whichstimulate the greatest T cell response for assessment in an animal or ina human. In another embodiment, one or more of the vaccine compositionsproducing a stimulated T cell response greater than a pre-selected valueare selected for in vivo assessment. Alternatively, one or more vaccinecompositions having relatively high activity compared to the remainingvaccine compositions are selected for in vivo assessment.

In a further embodiment, the invention relates to a method for assessingthe ability of a vaccine composition comprising one or more antigens orone or more nucleic acid molecules encoding one or more antigens tostimulate a protective T cell response. The method comprises the stepsof: (1) contacting human antigen presenting cells in culture with thevaccine composition, thereby, if one or more of the antigens or nucleicacid molecules can be taken up and processed by the antigen presentingcells, producing one or more processed antigens; (2) contacting theantigen presenting cells with human T cells under conditions sufficientto produce a T cell response to one or more of the processed antigens,thereby producing a T cell response; (3) measuring the T cell response;and, if the T cell response is greater than a pre-selected value, (4)assessing the ability of the vaccine composition to stimulate aprotective T cell response in one or more animals, human subjects or acombination thereof. The pre-selected value of the T cell response is,typically, chosen to represent a vaccine composition which isparticularly active in stimulating a T cell response.

In another embodiment, the method of the invention comprises the stepsof: (1) contacting human antigen presenting cells in culture with thevaccine composition, whereby, if one or more of the antigens are takenup and processed by the antigen presenting cells, said antigen orantigens are processed by the antigen presenting cells, therebyproducing one or more processed antigens; (2) contacting the antigenpresenting cells of step (1) with human T cell clones which are specificfor an epitope within one or more of the antigens for a period of timesufficient for the human T cell clones to respond to one or more of theprocessed antigens; and (3) determining whether the human T cell clonesrespond to the processed antigen or antigens. If the T cell clonesrespond to the processed antigen or antigens, the method can,optionally, further include the step of assessing the vaccinecomposition in an animal or in a human.

A “processed antigen”, as the term is used herein, refers to one or moreepitopes derived from an antigen which are presented at the surface ofan antigen presenting cell in combination with MHC I or MHC II.

The present method assesses the ability of a candidate vaccinecomposition to provide in vitro an antigen to antigen presenting cellsin a manner which leads to processing and presentation of one or more Tcell epitopes at the surface of the antigen presenting cells incombination with MHC I or MHC II. This in vitro determination providesan efficient screen for selecting compositions for more time-consumingin vivo testing in animals or in humans. This in vivo testing can beperformed using methods which are well known in the art. For example,the vaccine composition can be administered to an animal or a human, andthe ability of the induced immune response, if any, to protect againstsubsequent challenge from the pathogen from which the antigen orantigens are derived can be determined. Alternatively, or in conjunctionwith such a determination, the ability of the vaccine composition toinduce in vivo the proliferation of T cells and/or antibodies whichrecognize one or more of the antigens can also be determined. Animalswhich can be used for in vivo testing include laboratory animals,domesticated animals and wild animals. Suitable examples includerodents, such as mice, hamsters, rats, guinea pigs and rabbits;primates, such as monkeys and apes; and domestic animals, such as dogs,cats, horses, chickens, cows and pigs.

The antigen presenting cells are contacted with the vaccine compositionin cell culture in a suitable culture medium, as is known in the art,and under suitable conditions, such as physiological pH, and at atemperature from about room temperature to about physiologicaltemperature, for a sufficient period of time for uptake and processingof the antigen by the antigen presenting cells. If the vaccine comprisesa nucleic acid molecule, the antigen presenting cells are contacted withthe vaccine composition for a sufficient amount of time for the antigenpresenting cells to take up and express the nucleic acid molecule andprocess the resulting antigen. Generally, the antigen presenting cellsare contacted with the vaccine composition for a period of severalhours, for example, from about 2 to about 12 hours. Following contactwith the vaccine composition, the antigen presenting cells are contactedwith the T cells for a sufficient period of time for activation of the Tcells and generation of a T cell effector response. Generally, thisprocess requires several hours, for example, from about 2 to about 12hours. Preferably, the APCs are contacted with the vaccine compositionfor a sufficient period time for antigen or nucleic acid moleculeuptake, and then washed and placed in fresh media prior to addition ofthe T cells. Alternatively, the antigen presenting cells can becontacted with the vaccine composition and the T cells simultaneously orwithin a relatively short time interval. In this embodiment, the antigenpresenting cells are contacted with the vaccine composition and the Tcells for a sufficient amount of time for antigen processing andgeneration of a T cell response. Typically, such a process requires fromabout 4 to about 24 hours.

The vaccine composition, preferably, comprises at least one antigen, ora nucleic acid encoding at least one antigen, which is a protein or apeptide which comprises one or more T cell epitopes, such as one or moreCD8⁺ T cell epitopes, one or more CD4⁺ T cell epitopes or a combinationthereof. Preferably, the T cells are specific for a particular epitopepresent within the antigen. More preferably, the T cells are T cellclones derived from a single precursor T cell. In a particularlypreferred embodiment, the T cells are human T cell clones.

In one embodiment, the epitope is a CD4⁺ T cell epitope and the T cellsare CD4⁺ T cells. As discussed above, the effector functions of CD4⁺ Tcells include releasing cytokines and stimulating B cells to becomeantibody-producing cells. Thus, in this embodiment, the extent of the Tcell response to the antigen presenting cells can be determined bymeasuring T cell proliferation, the production of one or more cytokinesor the stimulation of antibody production by B cells. Greater levels ofT cell proliferation, antibody production or cytokine production wouldbe expected to correlate with greater immunogenicity and potentialefficacy of the vaccine composition.

In another embodiment, the epitope is a CD8 epitope and the T cells areCD8⁺ T cells. As discussed above, the effector functions of CD8⁺ T cellsinclude lysis of antigen presenting cells and release of cytokines.Therefore, the extent of CD8⁺ T cell response to the antigen presentingcells can be determined using an assay for cell lysis or by measuringthe production of one or more cytokines. The CD8⁺ T cell response canalso be measured by measuring the extent of release of one or morecytokines. In general, it is expected that greater cell lysis activityor cytokine release will correlate with greater immunogenicity.

The antigen presenting cells can be selected from among any suitablecells which are potentially capable of taking up the antigen, such as anatural, purified or recombinant protein, or a nucleic acid moleculeencoding the antigen, and presenting a peptide epitope derived from theantigen at the cell surface in combination with MHC I or MHC II. Forexample, when the epitope is a CD4 epitope, cells expressing MHC IImolecules can be used. Such cells include macrophages, dendritic cellsand B cells. When the epitope is a CD8⁺ T cell epitope, the antigenpresenting cells can be selected from among any cells which expressMHC 1. In preferred embodiments, the antigen-presenting cells areprofessional antigen-presenting cells, such as macrophages, dendriticcells and B cells. The antigen presenting cells can be, for example,recombinant cells expressing heterologous MHC molecules. In a preferredembodiment, the antigen presenting cells are human cells. The antigenpresenting cells present the proper MHC molecules and are, preferably,at least partially HLA matched with the T cells. More preferably, theAPCs are autologous cells, that is, cells derived from the same donor asthe T cells.

In one embodiment, the T cells are clones which are specific for aparticular epitope, and the vaccine composition includes at least oneantigen which comprises the epitope or at least one nucleic acidmolecule encoding at least one antigen which comprises the epitope. Inthis embodiment, response of the epitope-specific T cell clones toantigen-presenting cells which have been contacted with the experimentalvaccine composition indicates that the vaccine composition is able toeffect the presentation of the epitope on the surface of theantigen-presenting cells in combination with an MHC I or MHC IImolecule.

Epitope-specific T cell clones can be generated using methods which aregenerally known in the art (see, for example, Fathman, et al., in Paul,ed., Fundamental Immunology, second edition, Raven Press (1989), Chapter30, the contents of which are hereby incorporated by reference in theirentirety). The isolation of epitope-specific T cell clones is based on Tcell biology. Generally, an animal, such as a mouse, is immunized with apreparation of antigens (a bacterial lysate, or a purified protein) oris infected with a virus, such as a wild type virus or a recombinantvirus containing heterologous genes encoding one or more proteins from apathogenic microorganism, such as a virus. The animal is then sacrificedand the peripheral blood mononuclear cells (PBMC: includes T cells, Bcells, monocytes), spleen and lymph nodes are isolated. The isolatedcells are then cultured in media containing a defined component of theoriginal antigenic preparation, often a recombinant or purified protein,and the essential T cell growth factor interleukin-2 (IL-2). The only Tcells which will proliferate are those which recognize MHC/epitopecomplex in which the epitope is derived from the antigenic preparation.These cells become activated and proliferate while the unactivated cellsbegin to die. The cultures are maintained for several weeks, with themedia containing antigen and IL-2 being periodically replaced.Eventually, clusters of living and dividing cells (a T cell line) can beobserved in some of the cultures.

The proliferating cells are generally not clonal at this point and areof limited use for assaying epitope specific T cell responses. The Tcell line is, preferably, cloned through a process referred to aslimiting dilution. In this method, PBMC are isolated from, for example,a mouse of the same strain as the original mouse used to isolate the Tcell line. These cells, called antigen presenting cells, will serve as asource of MHC proteins and will present the MHC:peptide complex to the Tcell line. The T cell line is diluted to a concentration of about 1 to 5T cells/mL in a suspension of APCs that contains the antigen of interestand IL-2. This suspension is then transferred into, for example, roundor “v”-bottom 96 well microtitre plates, so that each well contains, onaverage, no more than 1 T cell. The cultures are maintained for severalweeks and a clone can grow out of one or more cultures.

The cells isolated by limiting dilution are the progeny of a single cellthat expresses only one T cell receptor, and the clone is thusepitope-specific. However, in a situation in which the cloning procedureuses whole proteins or viruses, a single protein may contain manyepitopes and the precise epitope will remain unknown. The epitope can beidentified using a collection of overlapping synthetic peptides thatspan the entire amino acid sequence of the antigenic protein. Thesepeptides can be used to stimulate proliferation or cytokine secretion ina direct stimulation assay, or they may be used as competitiveinhibitors to block activation of the T cell clone by the antigenicprotein.

Human T cell clones can also be isolated. Generally, these clones areisolated from individuals who have had an infection, for example,influenza, HIV or Dengue, or have been exposed to antigens in nature orby injection and have T cells that specifically respond to thoseantigens. These antigens are called “recall antigens” and includetetanus toxoid and Candida albicans extract. Human T cell clones areisolated from the PBMC.

The T-cell response to APCs treated with the test vaccine compositioncan be determined using a variety of assays which are known in the art.Several examples are taught by Fathman, et al., supra. For example, Tcell proliferation can be measured using methods known in the art. Inone embodiment, the epitope-specific T cells are mixed with irradiatedantigen presenting cells and the test vaccine composition and cultured.The cells are cultured for a period of a few days to allow presentationof the epitope by the APCs and activation of the T cells. T cellproliferation is then assessed by monitoring the incorporation of³H-thymidine into newly synthesized DNA. The APCs do not incorporate³H-thymidine because they have been irradiated. Alternative methods forassessing proliferation that do not use radioisotopes are also known.

T cell response can also be determined by determining if one or morecytokines is released by the T cells. For this assay, APCs and the testvaccine composition are mixed and cultured. Either simultaneously orafter a period of time sufficient for uptake and processing of anantigen within the vaccine composition by the APCs, T cells are added tothe culture. After a period of time sufficient to allow activation ofthe T cells, growth of the culture is stopped, for example, by freezing.Freezing the culture lyses the cells and releases cytokines that havenot yet been secreted into the culture medium. The presence or absenceof cytokine in the culture medium can then be determined using knownmethods. Optionally, the amount of one or more cytokines in the culturemedium can be determined. For example, cytokines in the culturesupernatant and the cells can be measured using a bioassay, in whichcell lines that proliferate only when stimulated with a particularcytokine (indicator cells) are cultured in media that is supplementedwith an aliquot of the cytokine-containing culture media. The culture ismaintained, typically, for 10-18 hours and ³H-thymidine is added. Afteran additional 6-10 hours, new DNA synthesis is measured by determiningthe amount of ³H incorporated into the cellular DNA. Any cytokine whichis produced by the T cells upon activation can be measured. Examples ofcytokines which can be determined include interferon-γ andinterleukin-2.

In another embodiment, cytokine production is measured using anenzyme-linked immunosorbent assay (ELISA), for example, using reagentswhich are commercially available as kits. In this assay, an immobilizedantibody is used to specifically capture a particular cytokine from thecytokine containing culture supernatant. Unbound proteins are washedaway, and the amount of bound cytokine is determined by binding asecond, labeled, antibody to the captured cytokine. This assay isquantitative and more specific than bioassays. Alternatively, cytokinemRNA levels can be quantitated using the polymerase chain reaction.Cytokine production can also be determined by staining producer T cellswith labeled antibodies specific for the cytokine.

In another embodiment, the T cells are CD8⁺ T cells and the response ismeasured by determining whether the T cells lyse the APCs which havebeen treated with the test vaccine composition. In one embodiment, theAPCs are transformed peripheral blood lymphocyte cell lines (B-LCL)which have been incubated with ⁵¹CrO₄ ²⁻. The resulting ⁵¹Cr-labeledPBLs are thoroughly washed, incubated with the test vaccine compositionand then exposed to the antigen-specific CD8⁺ T cells. After incubatingfor a sufficient period of time for epitope presentation by the B-LCLsand T cell activation, the extent of ⁵¹Cr release into the culturemedium is determined. The amount of ⁵¹Cr released correlates with theextent of lysis of the B-LCLs.

The production of a T cell response can, generally, be determined bycomparing the result achieved with the vaccine composition to a suitablecontrol, as is known in the art. For example, in the ⁵¹Cr release assaydiscussed above, the amount of ⁵¹Cr released when the B-LCLs are treatedwith the CD8⁺ T cells can be compared to the amount released when theB-LCLs are treated with vehicle alone, referred to as the backgroundrelease. Significantly (measurably) greater ⁵¹Cr release in the presenceof the T cells is indicative of a T cell response. In the cytokineproduction assay, cytokine production by the T cells in the presence ofAPCs treated with the vaccine composition can be compared to cytokineproduction by the T cells in the absence of APCs, or in the presence ofuntreated APCs. Greater cytokine production in the presence of treatedAPCs is indicative of a T cell response.

The test vaccine composition comprises one or more antigens or one ormore nucleic acid molecules which encode one or more antigens. Thevaccine composition can be any of the types of vaccine compositionswhich are known in the art. For example, the vaccine composition cancomprise an attenuated pathogen, such as a weakened bacterial strain orvirus, or a killed pathogen, such as a killed bacterial strain or akilled virus. The vaccine composition can also comprise a portion of apathogen, for example, a viral coat or bacterial membrane. In anotherembodiment, the vaccine composition comprises one or more proteinsderived from a pathogen, for example, a protein which has been purifiedor partially purified from the pathogen, or a recombinant proteinproduced by a recombinant organism which expresses a gene derived fromthe pathogen which encodes the protein. Examples of suitable hostorganisms for the production of recombinant peptides and proteins areknown in the art and include E. Coli. The vaccine composition can alsoinclude one or more fragments of a protein or proteins derived frompathogen. Such protein fragments include peptides which are synthesizedor recombinantly produced.

In another embodiment, the test vaccine composition includes one or moreproteins, or fragments thereof, which are produced by a particular typeof tumor cell. Preferably, the protein is unique to the tumor cell,i.e., not present in or on healthy cells, or is expressed in greaterquantity by the tumor cell than by healthy cells. The protein can be,for example, a protein found on the surface of the tumor cell. Theprotein(s) can be derived from the tumor cells, for example, isolatedand purified or partially purified from cultured tumor cells. The tumorcell protein(s), or a fragment or fragments thereof, can also beproduced recombinantly.

In another embodiment, the vaccine composition comprises a nucleic acidmolecule which encodes a protein or a fragment thereof, derived from apathogen or a tumor cell as discussed above. For example, the vaccinecomposition can comprise so-called “naked DNA”. The nucleic acidmolecule can also be contained within a suitable vector, such as arecombinant virus, such as vaccinia virus, adenovirus, orf virus,fowlpox virus, herpes virus, varicella virus, papilloma virus, SV40,retroviruses, baculovirus and poliomyelitis virus. The vector can alsobe a bacterium, such as salmonella, BCG or E. coli. The nucleic acid canalso be present in a liposome or another suitable vector, such as areknown in the art.

As discussed above, the present invention enables the rapid assessmentand comparison of a large number of potential vaccine compositions. Forany given disease or pathogen, for example, a variety of antigens can beassessed. For example, a set of vaccine compositions which each includedifferent antigens or portions of antigens from a particular pathogencan be compared. Further, for a given antigen, set of antigens, ornucleic acid molecule encoding such antigen(s), a variety offormulations can be assessed. For example, a set of vaccine compositionsincluding the same antigen or antigens, but different vectors,adjuvants, concentrations, vehicles or excipients can be compared todetermine the conditions necessary for optimal efficacy.

The invention will now be further and specifically described in thefollowing examples.

EXAMPLES

Materials and Methods

Viruses

Influenza A viruses A/Puerto Rico/8/34 (H1N1) and A/Japan/305/57 (H2N2)were obtained from the Division of Virology, Bureau of Biologics, Foodand Drug Administration, Bethesda, Md. A/Johannesburg/94 (H3N2) wasobtained from David Burt (Pasteur Merieux Connaught, Toronto, Ontario,Canada). Influenza A viruses were propagated in 10-day-old, embryonatedchicken eggs. Infected allantoic fluids were harvested 2 days afterinfection, aliquoted, and stored at −80° C. until use. Recombinantvaccinia viruses containing the genes coding for influenza A viralproteins HA, NA, M1, M2, PB 1, PB2, PA, NS 1, and NS2 and thenucleoprotein (NP) were obtained from B. Moss. Each of these was derivedfrom the A/PR/8/34 influenza A virus strain, except for NS 1, which wasderived from A/Udorn/72. They were constructed and propagated aspreviously described (Smith et al., Virology 160: 336-345 (1987)). Arecombinant vaccinia virus which expressed segmented portions of the NPwas obtained from J. Bennink and L. Eisenlohr.

Human PBMC

PBMC specimens were obtained from normal, healthy donors. Most of thedonors whose PBMC were tested had convincing evidence of influenza Avirus-specific CTL activity in bulk culture. PBMC were purified byFicoll-Hypaque density gradient centrifugation (A. Boyam, Scand. J.Clin. Lab. Invest. 21: 77-89 (1968)). Cells were resuspended at 2×10⁷/mLin RPMI 1640 with 20% fetal bovine serum (FBS) (Sigma) and 10% dimethylsulfoxide and cryopreserved until use. The HLA alleles of donor 1 wereA2.1, A11, B18, B27, Cw1, Cw7, DR1, DQw1, DQw3, DRw52, and DRw53. HLAtyping was performed in the HLA typing laboratory at the University ofMassachusetts Medical Center.

Bulk Cultures of PBMC

Responder PBMC were suspended at 10⁶/mL in AIM-V medium (Gibco BRL,Grand Island, N.Y.) containing 10% human AB serum (NABI, Boca Raton,Fla.), penicillin-streptomycin, glutamine, and HEPES in a 70-mL flask(Falcon). Stimulators were infected with A/PR/8/34 at a multiplicity ofinfection (MOI) of 15 for 1.5 h at 37° C. in 1 mL of phosphate-bufferedsaline containing 0.1% bovine serum albumin and then added to respondersin a flask at a stimulator-responder ratio of 1:10. On day 7 of culture,cells were either cloned by limiting dilution as described below orrestimulated with gamma-irradiated (3,000 rads) autologous PBMC infectedwith A/PR/8/34 at an MOI of 15 for 1.5 h in 1 mL of phosphate-bufferedsaline containing 0.1% bovine serum albumin, added at astimulator-responder ration of 1:10 in fresh medium containing 10% humanAB serum and 20 U of interleukin-2 (IL-2) (Collaborative BiomedicalProducts, Bedford, Mass.). Restimulated cells were either cloned bylimiting dilution or assayed for cytolytic activity 7 days later.

CTL Clones

Influenza virus-specific CTL clones were established by using alimiting-dilution technique as previously described (Kurane et al., J.Exp. Med. 170: 763-775 (1989)). PBMC which had been stimulated in bulkculture for 7 or 14 days were collected and plated at a concentration of3, 10, or 30 cells per well in 96-well round-bottom microtiter plates in100 μL of AIM-V medium containing 10% FBS, 20 U of IL-2, a 1:1,000dilution of anti-CD8 monoclonal antibody 12F6 (obtained from JohnsonWong), and 10⁵ gamma-irradiated allogeneic PBMC/well. On day 7, 50 μL offresh medium with FBS (Sigma Immunochemicals, St. Louis, Mo.) and IL-2were added, and on day 14, fresh medium with 10⁵ gamma-irradiatedallogeneic PBMC/well and a 1:1,000 dilution of the anti-CD8 monoclonalantibody were added. Growing cells were assayed for cytolytic activityon days 21 and 28. Cells from wells with influenza A virus-specificcytolytic activity were expanded to 48-well plates.

Preparation of Target Cells

Autologous lymphoblastoid cell lines (B-LCLs) were established byculturing with Epstein-Barr virus in 24-well plates as previouslydescribed (Green et al., J. Virol. 67: 5962-5967 (1993)). B-LCL wereinfected with recombinant vaccinia viruses at an MOI of 20:1 for 1.5 hat 37° C. The cells were then diluted in 1 mL of medium and furtherincubated for 12 to 16 h. Other B-LCL were infected with A/PR/8/34,A/Japan/305/57, or A/Johannesburg/94 in 1 mL of medium for 12 to 16 h.These infected target cells were labeled with 0.25 mCi of ⁵¹Cr for 60min at 37° C. After four washes, the target cells were counted anddiluted to 2×10⁴/mL for use in the cytotoxicity assay. The partiallyHLA-matched allogeneic target cells used in the assays were B-LCLproduced in our laboratory form the HLA-typed PBMC of unrelated donorsor were obtained from the National Institute of General Medical SciencesHuman Genetic Mutant Cell Repository or the American Society forHistocompatibility and Immunogenetics Cell Bank and Repository.

Cytotoxicity Assays

Cytotoxicity assays were performed with 96-well round-bottom plates.Effector cells in 100 μL of RPMI 1640 medium containing 10% FBS wereadded to 2×10⁵ ⁵¹Cr-labeled target cells in 100 μL at aneffector-to-target (E-T) ratio of 10:1. Plates were centrifuged at 200×gfor 5 min and incubated for 4 to 5 h at 37° C. Supernatant fluids wereharvested by using the supernatant collection system (SkatronInstruments, Sterling, Va.), and ⁵¹Cr content was measured in a gammacounter. Percent specific ⁵¹Cr release was calculated with the followingformula: (cpm experimental release−cpm spontaneous release)/(cpm maximumrelease−cpm spontaneous release)×100. All assays were performed intriplicate, and the results were calculated from the average of thetriplicate wells.

EXAMPLE 1 In Vitro Evaluation of Influenza Vaccine Compositions

This Example was designed to evaluate whether proper formulation of aninfluenza virus comprising a formalin-inactivated detergent-disruptedvirus can lead to a CD8⁺ cytotoxic T cell response.

The HA-Specific CD8⁺ cytotoxic T cell clone described in Example 1 wasincubated with autologous B-LCL cells which had been treated with one ofthe following vaccine formulations:

-   -   1. live influenza virus (H1N1, A/PR/8/34, A/Texas/9I)    -   2. iscomatrix alone    -   3. formalin-inactivated A/Texas/H1N1 virus; and    -   4. formalin-inactivated A/Texas/H1N1 virus formulated with        Iscoms.

The results are shown in Table 1, which provides cytolysis as a percentof total APCs and the background ⁵¹Cr release. The data show that theCD8⁺ clone recognized APC infected with live flu virus or a recombinantvaccinia virus. This CD8⁺ CTL clone, however, did not lyse APC pulsedwith the inactivated A/Texas/H1N1 virus unless it was formulated with anadjuvant carrier. Formulation with Iscoms enabled processing of thevaccine for CD8⁺ CTL recognition. TABLE 1 Percent lysis of B-LCLstreated with vaccine formulations by HA- specific cytotoxic T cell clonePR/8 A/Tx A/Tx A/Tx Flu - virus virus Iscomatrix vaccine Iscoms CD8 +36.1% 19.5% −3.5% −2.2% 86.9% clone min/max 12.8% 12.1% 17.4% 41.3%44.6%

EXAMPLE 2 In Vitro Evaluation of Recombinant Protein VaccineFormulations

A human CD8⁺ cytotoxic T cell clone that recognizes amino acids 122-130of the Influenza A NS1 protein is disclosed in U.S. Pat. No. 5,766,601,the teachings of which are incorporated herein by reference in theirentirety. This T cell clone was incubated with autologous ⁵¹Cr-labeledB-LCL treated with (1) a synthetic peptide based on NS 1 (aa 122-130);(2) recombinant NS 1 protein; or (3) recombinant NS 1 protein formulatedas an Iscom. Controls were also established using uninfected B-LCL andB-LCL incubated with Iscoms only.

The results of this study are presented in Table 2. The CD8⁺ cytoxic Tcell clone lysed APC that were treated with the recombinant NS 1 aa122-130 peptide and the recombinant NS 1 protein/Iscom formulations. TheCTL clone did not lyse APC treated with the recombinant protein alone oreither of the control cells. TABLE 2 Percent lysis of autologous B-LCLtreated with the indicated formulations by autologous NS1 aa 122-130specific CD8⁺ cytotoxic T cell clone NS1 aa NS1 protein Iscom uninfected122-130 a NS1 Iscom b, c d CD8⁺ −6.2% 88.4 −0.7% 19.6% −11.0 clonemin/max 17.8% 19.6 −17.3% 29.9% 21.3%a Peptide used at 25 μg/mL;b Protein used at 35 μg/mL;c Saponin used at 100 μg/mL;d Saponin used at 70 μg/mL

EXAMPLE 3 In Vitro Evaluation of HIV-1 Vaccine Compositions

A human HIV-1 specific CD8⁺ cytotoxic T cell clone was prepared asdescribed by Littaua et al., J. Virol. 65: 4051-4056 (1991), theteachings of which are hereby incorporated by reference in theirentirety. The ability of this clone to recognize autologous APCs pulsedwith (1) recombinant HIV-1 p24 protein alone; (2) HIV-1 p24 in an Iscomformulation; or (3) a recombinant vaccinia virus containing the HIV-1p24 gene was determined.

The results are presented in Table 3, which shows that APCs treated withthe recombinant vaccinia virus are significantly lysed by the T cellclone. APCs treated with the p24/Iscoms complex are also recognized bythe T cell clone, but to a lesser extent. The T cell clone did notrecognize B-LCLs pulsed with the recombinant p24 protein alone. TABLE 3Lysis of B-LCL treated with indicated formulations by human HIV-1specific CD8⁺ cytotoxic T cell clone Iscoms + p24 at Iscom Media Vac/p24100 μg 50 μg 25 μg 100 μg 37.5 11.4 20.2 −0.1 −2.6 0.3

EXAMPLE 5 In Vivo Evaluation of Influenza Virus Compositions

Fifty-five healthy adults from 18 to 45 years old were enrolled in 5study groups of 11 participants each: 1. Fluzone; 2. Flu-Iscom (75 μg);3. Flu-Iscom (50 μg); 4. Flu-Iscomatrix (75 μg); and 5. Flu-Iscomatrix(50 μg). Cytotoxic T cell activity in the peripheral blood lymphocytesof the subjects was determined on days 0, 14 and 56 following a singleimmunization with trivalent vaccine. Peripheral blood lymphocytes ateach time point for each subject were tested in the same assay forkilling of virus-infected autologous target cells (Epstein-Barr virustransformed B cells) at various E:T ratios (90, 30 and 10). Responderswere those subjects which showed a significant increase in killing ofgreater than 5% compared with the percent net lysis at time 0 at two ormore effector:target ratios. The results are presented in Table 4. TABLE4 Fluzone Virus alone Flu-Iscom Flu-Iscom Flu MTRX Flu MTRX Strain 50/4575/45 50/45 75/45 50/45 H1 0/11 7/11 5/11 1/11 5/11 H3 1/11 3/11 7/113/11 5/11 B 5/11 6/11 5/11 6/11 7/11

These results show that the effect of the adjuvant was significant forvirus strains H1 and H3, while results were similar for strain B in thepresence and absence of an adjuvant. FIGS. 1A and 1B also present dataillustrating the increase in net lysis compared to day 0 for strainA/Texas at days 14 and 56, respectively. Similar data for strainA/Johannesburg are illustrated in FIGS. 2A and 2B.

Equivalents

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the spirit and scope of theinvention as defined by the appended claims. Those skilled in the artwill recognize or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described specifically herein. Such equivalents are intendedto be encompassed in the scope of the claims.

1. A method for assessing the ability of a vaccine composition tostimulate a T cell response, wherein the vaccine composition comprisesone or more antigens or one or more nucleic acid molecules encoding oneor more antigens, said method comprising the steps of: (a) contactingantigen presenting cells in culture with the vaccine composition,thereby, if one or more of the antigens or nucleic acid molecules aretaken up and processed by the antigen presenting cells, producing one ormore processed antigens; (b) contacting the antigen presenting cellswith T cells under conditions sufficient for the T cells to respond tothe processed antigen; and (c) determining whether the T cells respondto the processed antigen; whereby, if the T cells respond to theprocessed antigen, the vaccine composition is capable of stimulating a Tcell response; and if the vaccine composition is capable of stimulatinga T cell response; (d) assessing the vaccine composition in one or moreanimals or human subjects.
 2. The method of claim 1 wherein the T cellsare human T cells.
 3. The method of claim 2 wherein the antigenpresenting cells are human antigen presenting cells.
 4. The method ofclaim 2 wherein the T cells are CD8⁺ T cells.
 5. The method of claim 2wherein the T cells are CD4⁺ T cells.
 6. The method of claim 2 whereinthe antigen presenting cells are selected from the group consisting ofmacrophages, dendritic cells and B cells.
 7. The method of claim 2wherein the T cell response to the antigen is the release of one or morecytokines or lysis of the antigen presenting cells.
 8. The method ofclaim 3 wherein the T cell response to the antigen which is measured isrelease of one or more cytokines or stimulated formation of antibodiesby B cells.
 9. The method of claim 1 wherein the antigen comprises a Tcell epitope.
 10. The method of claim 9 wherein the T cells are T cellclones.
 11. A method for selecting one or more vaccine compositions fromamong a group consisting of two or more vaccine compositions forassessment in an animal or in a human, said vaccine compositions eachcomprising one or more antigens or one or more nucleic acid moleculesencoding one or more antigens, said method comprising the steps of: (a)contacting antigen presenting cells in culture with a vaccinecomposition selected from among said group of vaccine compositions,thereby, if one or more of the antigens or nucleic acid molecules aretaken up and processed by the antigen presenting cells, producing one ormore processed antigens; (b) contacting the antigen presenting cellswith T cells under conditions sufficient for the T cells to respond toone or more of the processed antigens; (c) determining whether the Tcells respond to one or more of the processed antigens; whereby if the Tcells respond to one or more of the processed antigens, then the vaccinecomposition stimulates a T cell response; (d) repeating steps (a), (b)and (c) with each additional vaccine composition in the group, therebydetermining whether each vaccine composition stimulates a T cellresponse; and, if one or more of the vaccine compositions stimulates a Tcell response (e) selecting at least one vaccine composition whichstimulates a T cell response for assessment in one or more animals orhuman subjects.
 12. The method of claim 11 wherein the T cells andantigen presenting cells are human cells.
 13. The method of claim 11wherein the T cells are human T cell clones.
 14. A method for selectingone or more vaccine compositions from among a group consisting of two ormore vaccine compositions for in vivo assessment in one or more animalsor human subjects, said vaccine compositions each comprising one or moreantigens or one or more nucleic acid molecules encoding one or moreantigens, said method comprising the steps of: (a) contacting antigenpresenting cells in culture with a vaccine composition selected fromamong said group of vaccine compositions, thereby, if one or more of theantigens or nucleic acid molecules are taken up and processed by theantigen presenting cells, producing one or more processed antigens; (b)contacting the antigen presenting cells with T cells under conditionssufficient to produce a T cell response to one or more of the processedantigens, thereby producing a vaccine composition-stimulated T cellresponse; (c) measuring the vaccine composition-stimulated T cellresponse; (d) repeating steps (a), (b) and (c) with each of theremaining vaccine compositions in the group, thereby identifying thevaccine composition or compositions which stimulate the greatest T cellresponse; (e) selecting the vaccine composition or compositions whichstimulate the greatest T cell response for in vivo assessment in one ormore animals or human subjects.
 15. The method of claim 14 wherein the Tcells are human T cells and the antigen presenting cells are humanantigen presenting cells.
 16. The method of claim 15 wherein the T cellsare human T cell clones.
 17. The method of claim 16 wherein the T cellsare CD8⁺ T cell clones or CD4⁺ T cell clones.
 18. A method for assessingthe ability of a vaccine composition comprising one or more antigens orone or more nucleic acid molecules encoding one or more antigens tostimulate a human T cell response, said method comprising the steps of:(a) contacting human antigen presenting cells in culture with thevaccine composition, thereby, if one or more of the antigens or nucleicacid molecules can be taken up and processed by the antigen presentingcells, producing one or more processed antigens; (b) contacting theantigen presenting cells with human T cells under conditions sufficientto produce a T cell response to one or more of the processed antigens,thereby producing a T cell response; (c) measuring the T cell response;and if the T cell response is greater than a pre-selected value, (d)assessing the ability of the vaccine composition to stimulate aprotective T cell response in one or more animals or human subjects. 19.The method of claim 18 wherein the T cells are CD8⁺ T cell clones orCD4⁺ T cell clones.
 20. The method of claim 18 wherein the antigenpresenting cells are autologous cells.